Research efforts in chiral chemistry and enantioselective production technologies continue to increase as the U.S. Food and Drug Administration and the European Committee for Proprietary Medicinal Products push pharmaceutical companies to market single-isomer drugs. Manufacturers are now required to characterize each enantiomer in all drugs that are proposed to be marketed as a mixture. The production of new racemates (mixtures of enantiomers) has ceased to be a viable commercial option with these new guidelines. The push to market chirally-pure drugs is being driven by the realization that often only one enantiomer of a racemate is responsible for efficacy, while the nonefficacious enantiomer has at best no physiological effect and at worst is responsible for adverse side effects. Thalidomide is a tragic example of a drug where efficacy resides in one enantiomer and the side effect, teratogenicity, resides in the other.
Among the enantioselective production technologies are kinetic resolution and asymmetric synthesis by biocatalysts, such as enzymes. Biocatalysis is becoming increasingly popular in drug and specialty chemical manufacturing, mainly due to its simplicity, versatility, and environmental acceptability. Among the more useful reactions by which chirality can be introduced are stereospecific hydrolysis and formation of ester and amide bonds, both of which reactions can be catalyzed by hydrolases. Of particular interest among the hydrolases are lipases, esterases, glycosidases, proteases, and amidases.
Many potential industrial applications for enzymes involve substrates, organic solvents and other reaction conditions that are never encountered in nature. Protein engineering can be used to change the properties of enzymes to supply the needs of pharmaceutical manufacturing. By carefully controlling in vitro mutation efficiencies and screening for enhanced catalytic properties over multiple generations, new enzymes can be developed that are hundreds of times more active than the natural enzymes in chemical process environments.
However, further exploration of this technology is severely limited by the current need to employ time-consuming liquid-phase assays for screening mutant libraries. Despite recent advances by pharmaceutical and biotechnology firms to accelerate their drug discovery efforts by developing automated liquid-phase screening systems, screening bottlenecks remain. For example, conventional 96-well microtiter plates, with 100-200 .mu.l/well, are currently used in almost all screening. This volume results in less throughput than is desired.
Improvements in assay miniaturization would allow acceleration in the rate of screening, reduction in the cost per assay, and conservation of compounds that are either expensive or difficult to synthesize and purify. Thus, there exists in the art a need to develop a high throughput solid phase screening system, reducing effective assay volumes to 100 nanoliters (nl) or less.
In 1994, KAIROS Scientific Inc. developed and commercialized a PC-based ColonyImager capable of determining the absorption spectra of several hundred cell colonies on a single Petri dish (Youvan, Nature 369: 79-80, 1994). This device has proved very useful in the isolation of mutants of Rhodobacter capsulatus expressing natural pigments such as carotenoids and bacteriochlorophyll. In concert with directed evolution techniques, the ColonyImager was used to screen libraries of mutagenized photosynthetic bacteria for light-harvesting proteins that were undergoing directed evolution through repeated cycles of combinatorial mutagenesis. However, the throughput of the Colonylmager is limited to a few (.about.10) colonies per square centimeter.
Accordingly, the inventors have determined that it would be highly useful to automatically determine the spectroscopic characteristics, particularly over time, of a large number of cellular colonies expressing variants of a selected enzyme. The present invention provides a system and methods for accomplishing this end.